α-Crystallin, a major lens protein having homology with small heat shock proteins, prevents aggregation of other proteins like a molecular chaperone. The inventors had earlier shown that α-crystallin can prevent photo-aggregation of γ-crystallin, which may have relevance in cataractogenesis.
By using various non-thermal modes of aggregation, it was shown that chaperone-like activity of α-crystallin is temperature-dependent. A structural perturbation above 30° C. enhances this activity severalfold.
In order to probe the molecular mechanism of the chaperone-like activity and its enhancement upon structural perturbation, the inventors have studied α-crystallin and its constituent subunits The recent study of the αA and αB heteroaggregates showed that, despite high sequence homology, these proteins differ in their stability, chaperone-like activity, and the temperature dapendence of this activity. This study also indicated different roles for the two proteins in that α-crystallin heteroaggregate in the eye lens and as separate proteins in non-lenticular tissues.
Several investigators have introduced mutations in αA and αB crystallins to gain an insight into the structure-function relation. Derham and Harding in their recent review list about 30 site-directed mutations from different laboratories. These mutations either result in some decrease or no change in protective ability. It is interesting to note that point mutations in both αA and αB crystallin, R116C and R120G, respectively, result in significant loss of activity and are associated with human diseases.
Human αA and αB crystallins are coded by three exons and are thought to have arisen due to gene duplication. They share high sequence homology with the small heat shock proteins, which are found in all organisms, from prokaryotes to humans. αA and αB crystallins are constitutively expressed during normal growth and development. αA crystallin is expressed predominantly in the eye lens with small amounts being present in the spleen and thymus, whereas αA crystallin is expressed not only in the eye lens, but also in several other tissues such as the heart, skeletal muscle, placenta, lung, and kidney.
The main function of these proteins in the lens appears to provide transparency and prevent precipitation by binding to other aggregation-prone proteins. In the lens, αA and αB crystallins exist as heteroaggregates of approximately 800 kDa. Both the recombinant αA and αB crystallins exist as high molecular mass oligomeric proteins of approximately 640 and 620 kDa, respectively. The size of these proteins can vary a little depending on the pH and ionic strength, and they differ in structure, function, tissue expression, and abnormal deposition in disease.
αB crystallin has a heat shock element upstream to the gene and is induced during stress. Apart from maintaining lens transparency, its in vivo functions include interaction with intermediate filaments and regulation of cytomorphological rearrangements during development. αB crystallin is hyperexpressed in neurological disorders such as Alzheimer's disease, Creutzfeldt-Jacob disease, and Parkinson's disease.
The charge C-terminal domain is conserved in all the members of the small heat shock protein family, whereas the hydrophobic N-terminal domain is variable in length and sequence similarity. The N- and C-terminal domains are thought to form two structural domains with an exposed C-terminal extension.
To investigate the role of the N-terminal domains in the differential structural and functional properties of human αA and αB crystallins, the inventors herein swapped their N-terminal domains coded by exon 1. A unique XmnI restriction site at the beginning of the α-crystallin domain in a 20-nucleotide stretch in exon 2, with 100% sequence identity human αA and αB crystallin genes, has been used to create chimeric proteins αANBC and αBNAC. The inventors herein used biophysical methods to study the structural and functional properties of wild-type αA and αB crystallins as well as the chimeras in order to get an insight into the effect of swapping and the role of N-terminal domain in oligomerization and chaperone-like activity.